CRF analogs

ABSTRACT

Analogs of rCRF and oCRF are disclosed that can be administered to achieve a substantial elevation of ACTH, β-endorphin, β-lipotropin, other products of the pro-opiomelanocortin gene and corticosterone levels and/or a lowering of blood pressure over an extended period of time. One analog which has been found to be particularly potent is: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH 2 . In the analogs, one or more of the first five N-terminal residues may be deleted or may be substituted by a peptide up to 10 amino acids long and/or by an acylating agent containing up to 7 carbon atoms. A number of other substitutions may also be made throughout the chain. These analogs or pharmaceutically or veterinarily acceptable salts thereof, dispersed in a pharmaceutically or veterinarily acceptable liquid or solid carrier, can be administered to mammals, including humans. These analogs may also be used as stimulants to elevate mood and improve memory and learning, as well as diagnostically.

This invention was made with Government support under Grant No. AM-26741 awarded by the National Institutes of Health. The Government has certain rights in this invention.

This invention is directed to peptides and to methods for pharmaceutical treatment of mammals using such peptides. More specifically, the invention relates to the hentetracontapeptide CRF, to analogs of CRF, to pharmaceutical compositions containing CRF or such analogs and to methods of treatment of mammals using CRF or such analogs.

BACKGROUND OF THE INVENTION

Experimental and clinical observations have supported the concept that the hypothalamus plays a key role in the regulation of adenohypophysial corticotropic cells secretory functions. Over 25 years ago, Guillemin, Rosenberg and Saffran and Schally independently demonstrated the presence of factors in hypothalamus which would increase the rate of ACTH secretion by the pituitary gland incubated in vitro or maintained in an organ culture. None of the secretagogs characterized met the criteria expected of a physiologic corticotropin releasing factor (CRF) until ovine CRF (oCRF) was characterized in 1981 and, as disclosed in U.S. Pat. No. 4,415,558, was found to have the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH₂.

Sauvagine is a 40-residue, amidated generally similar peptide which was isolated from the skin of the South American frog Phyllomedusa sauvagei. It was characterized by Erspamer et al. and was described in Regulatory Peptides, Vol. 2 (1981), pp. 1-13. Sauvagine has the formula: pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gln-Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Thr-Ile-NH₂. Sauvagine and oCRF have been reported to have biological activity in lowering blood pressure in mammals and in stimulating the secretion of ACTH and β-endorphin.

SUMMARY OF THE INVENTION

Rat CRF(rCRF) has been isolated, purified and characterized as a hentetracontapeptide having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂. It may alternatively be referred to as rat Amunine. The formula of human CRF has apparently been determined to be the same as that of rCRF. Synthetic rCRF and oCRF stimulate ACTH and β-endorphin activities in vitro and in vivo and substantially lower blood pressure for an extended time period.

Analogs of these 41-residue CRF peptides having the following formula have at least substantially the same biological activity in the foregoing respects as the native peptides: Y-R₁ -R₂ -R₃ -R₄ -R₅ -Ile-Ser-R₈ -R₉ -leu-R₁₁ -R₁₂ -R₁₃ -leu-leu-Arg-R₁₇ -R₁₈ -R₁₉ -Glu-R₂₁ -R₂₂ -R₂₃ -R₂₄ -R₂₅ -R₂₆ -R₂₇ -R₂₈ -R₂₉ -Gln-ala-R₃₂ -R₃₃ -Asn-Arg-R₃₆ -R₃₇ -R₃₈ -R₃₉ -R₄₀ -R₄₁ -NH₂ wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R₁ is Ser, D-Ser or des R₁ ; R₂ is Gln, pGlu, Glu, D-pGlu or des R₂ ; R₃ is Glu, Gly, D-Tyr or des R₃ ; R₄ is Pro, D-Pro or des R₄ ; R₅ is Pro or desR₅ ; R₈, R₁₂, R₁₉ and R₂₄ are selected from the group consisting of leu, Ile, ala, Gly, Val, Nle, Phe and Gln; R₉ is Asp or Glu; R₁₁ is Thr or Ser; R₁₃ is His, Tyr or Glu; R₁₇ is Glu or Lys; R₁₈ is Val, Nle or Met; R₂₁ is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R₂₂ is ala, Thr, Asp or Glu; R₂₃ is Arg, Orn, Har or Lys; R₂₅ is Asp or Glu; R₂₆ is Gln, Asn or Lys; R₂₇ is leu, Ile, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R₂₈ is ala, Arg or Lys; R₂₉ is Gln or Glu, R₃₂ is His, Gly, Tyr or ala; R₃₃ is Ser, Asn, leu, Thr or ala; R₃₆ is Lys, Orn, Arg, Har or Leu; R₃₇ is leu or Tyr; R₃₈ is Met or leu; R₃₉ is Glu or Asp; R₄₀ is Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln; R₄₁ is ala, Ile, Gly, Val, Leu, Nle, Phe, Gln or des R₄₁, provided however that R₃ is D-Tyr or R₄ is D-Pro or R₂₈ is Arg or R₂₉ and/or R₄₀ is Glu or R₃₆ is either Arg or Har or R₃₇ is Tyr; or a nontoxic addition salt thereof.

Pharmaceutical compositions in accordance with the invention include such CRF analogs, or nontoxic addition salts thereof, dispersed in a pharmaceutically or veterinarily acceptable liquid or solid carrier. The administration of such peptides or pharmaceutically or veterinarily acceptable addition salts thereof to mammals, particularly humans, in accordance with the invention may be carried out for the regulation of secretion of ACTH, βendorphin, β-lipotropin, other products of the pro-opiomelanocortin gene and corticosterone and/or for the lowering of blood pressure and/or for affecting mood, behavioral and gastrointestinal functions and autonomic nervous system activities. Furthermore CRF analogs may be used for the evaluation of the status of pituitary, cardiovascular, gastrointestinal or central nervous system functions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The nomenclature used to define the peptides is that specified by Schroder and Lubke, "The Peptides", Academic Press (1965) wherein, in accordance with conventional representation, the amino group appears to the left and the carboxyl group to the right. The standard 3-letter abbreviations to identify the alpha-amino acid residues, and where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated, e.g. Ser=L-serine, Nle=L-norleucine, Nva=norvaline, Har=homoarginine, Orn=ornithine etc. In addition the following abbreviations are used: leu=either L-leucine or CαCH₃ -L-leucine (CML) and ala=either L-alanine or CαCH₃ -L-alanine(CMA).

The invention provides analogs of CRF having the following Formula (I): Y-R₁ -R₂ -R₃ -R₄ -R₅ -Ile-Ser-R₈ -R₉ -leu-R₁₁ -R₁₂ -R₁₃ -leu-leu-Arg-R₁₇ -R₁₈ -R₁₉ -Glu-R₂₁ -R₂₂ -R₂₃ -R₂₄ -R₂₅ -R₂₆ -R₂₇ -R₂₈ -R₂₉ -Gln-ala-R₃₂ -R₃₃ -Asn-Arg-R₃₆ -R₃₇ -R₃₈ -R₃₉ -R₄₀ -R₄₁ -NH₂ wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R₁ is Ser, D-Ser or des R₁ ; R₂ is Gln, pGlu, Glu, D-pGlu or des R₂ ; R₃ is Glu, Gly, D-Tyr or des R₃ ; R₄ is Pro, D-Pro or des R₄ ; R₅ is Pro or desR₅ ; R₈, R₁₂, R₁₉ and R₂₄ are selected from the group consisting of leu, Ile, ala, Gly, Val, Nle, Phe and Gln; R₉ is Asp or Glu; R₁₁ is Thr or Ser; R₁₃ is His, Tyr or Glu; R.sub. 17 is Glu or Lys; R₁₈ is Val, Nle or Met; R₂₁ is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R₂₂ is ala, Thr, Asp or Glu; R₂₃ is Arg, Orn, Har or Lys; R₂₅ is Asp or Glu; R₂₆ is Gln, Asn or Lys; R₂₇ is leu, Ile, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R₂₈ is ala, Arg or Lys; R₂₉ is Gln or Glu, R₃₂ is His, Gly, Tyr or ala; R₃₃ is Ser, Asn, leu, Thr or ala; R₃₆ is Lys, Orn, Arg, Har or Leu; R₃₇ is leu or Tyr; R₃₈ is Met or leu; R₃₉ is Glu or Asp; R₄₀ is Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln; R₄₁ is ala, Ile, Gly, Val, Leu, Nle, Phe, Gln or des R₄₁. However, in this formula, at least one and preferably at least two of the following substituents are present: R₃ is D-Tyr, R.sub. 4 is D-Pro, R₂₈ is Arg, R₂₉ and/or R₄₀ is Glu, R₃₆ is either Arg or Har and R₃₇ is Tyr. Most preferably, at least one of these two substituents is the residue Glu in the 29- or 40-position.

These analogs that have been synthesized are at least as potent as native CRF. These analogs preferably include residues having a high alpha-helical forming potential as follows: R₁ is Ser, R₂ is Gln or Glu, R₃ is Glu, R₄ and R₅ are Pro, R₈ is leu, R₁₁ is Thr, R₁₂ is Phe or leu, R₁₃ is His or Glu, R₁₇ is Glu, R₁₈ and R₂₁ are Met or Nle, R₁₉ and R₃₇ are leu, R₂₂ and R₄₁ are ala, R₂₃ is Lys, R₂₄ and R₂₈ are ala, R₂₅ and R₃₉ are Glu, R₂₆ is Gln, R₂₇ is Glu or leu, R₂₉ is Glu, R₃₂ is His or ala, R₃₃ is Ser or leu, R₃₈ is Leu and R₄₀ is Ile or Glu. One analog which has been found to be particularly potent is: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ and is hereinafter referred to as AHC (for alpha-helical CRF). It remains potent even if shortened at the N-terminal.

The peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution addition. Certain CRF analogs which do not include D-isomer residues or unnatural amino acid residues may also be synthesized by recently developed recombinant DNA techniques.

Synthesis by the use of recombinant DNA techniques, for purposes of this application, should be understood to include the suitable employment of a structural gene coding for the desired form of CRF analog. The synthetic CRF peptide may be obtained by transforming a microorganism using an expression vector including a promoter and operator together with such structural gene and causing such transformed microorganism to express the CRF peptide. A non-human animal may also be used to produce the CRF peptide by gene-farming using such a structural gene and the general techniques set forth in U.S. Pat. No. 4,276,282 issued June 30, 1981 or using microinjection of embryos as described in W083/01783 published May 26, 1983 and W082/04443 published Dec. 23, 1982. The synthetic CRF peptide is then suitably recovered from the animal by extraction from sera or the like.

Common to chemical syntheses of peptides is the protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups which will prevent a chemical reaction from occurring at that site until the group is ultimately removed. Usually also common is the protection of an alpha-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha-amino protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that, as a step in the synthesis, an intermediate compound is produced which includes each of the amino acid residues located in its desired sequence in the peptide chain with various of these residues having side-chain protecting groups.

Also considered to be within the scope of the present invention are intermediates of the Formula (II): X¹ -R₁ (X²)-R₂ (X⁴ or X⁵)-R₃ (X⁵ or X)-R₄ -R₅ -Ile-Ser(X²)-R₈ -R₉ (X⁵)-leu-R₁₁ (X²)-R₁₂ (X⁴)-R₁₃ (X or X⁵)-leu-leu-Arg(X³)-R₁₇ (X⁵ or X⁶)-R₁₈ -R₁₉ (X⁴)-Glu(X⁵)-R₂₁ -R₂₂ (X² or X⁵)-R₂₃ (X³ or X⁶)-R₂₄ (X⁴)-R₂₅ (X⁵)-R₂₆ (X⁴ or X⁶)-R₂₇ (X⁴ or X⁵)-R₂₈ (X³ or X⁶)-R₂₉ (X⁴ or X⁵)-Gln(X⁴)-ala-R₃₂ (X)-R₃₃ (X² or X⁴)-Asn(X⁴)-Arg(X³)-R₃₆ (X⁶)-R₃₇ (X)-R₃₈ -R₃₉ (X⁵)-R₄₀ (X² or X⁴ or X⁵)-R₄₁ (X⁴)-X⁷ wherein: the R-groups are as hereinbefore defined.

X¹ is either hydrogen or an α-amino protecting group. The α-amino protecting groups contemplated by X¹ are those known to be useful in the art in the step-wise synthesis of polypeptides. Among the classes of α-amino protecting groups covered by X¹ are (1) acyl-type protecting groups, such as formyl, acrylyl(Acr), benzoyl(Bz) and acetyl(Ac) which are preferably used only at the N-terminal; (2) aromatic urethan-type protecting groups, such as benzyloxycarbonyl(Z) and substituted Z, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as t-butyloxycarbonyl (BOC), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such as fluorenylmethyloxycarbonyl(FMOC), cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl; and (5) thiourethan-type protecting groups, such as phenylthiocarbonyl. The preferred α-amino protecting group is BOC.

X² is a protecting group for the hydroxyl group of Thr and Ser and is preferably selected from the class consisting of acetyl(Ac), benzoyl(Bz), tert-butyl, triphenylmethyl(trityl), tetrahydropyranyl, benzyl ether(Bzl) and 2,6-dichlorobenzyl (DCB). The most preferred protecting group is Bzl. X² can be hydrogen, which means there is no protecting group on the hydroxyl group.

X³ is a protecting group for the guanidino group of Arg or Har preferably selected from the class consisting of nitro, p-toluenesulfonyl(Tos), Z, adamantyloxycarbonyl and BOC, or is hydrogen. Tos is most preferred.

X⁴ is hydrogen or a protecting group, preferably xanthyl(Xan), for the amido group of Asn or Gln.

X⁵ is hydrogen or an ester-forming protecting group for the β- or γ-carboxyl group of Asp or Glu, preferably selected from the class consisting of benzyl, 2,6-dichlorobenzyl, methyl, ethyl and t-butyl ester. OBzl is most preferred.

X⁶ is hydrogen or a protecting group for the side chain amino substituent of Lys or Orn. Illustrative of suitable side chain amino protecting groups are Z, 2-chlorobenzyloxycarbonyl(2-Cl-Z), Tos, t-amyloxycarbonyl(Aoc), BOC and aromatic or aliphatic urethan-type protecting groups as specified hereinbefore.

When His is present, X is hydrogen or a protecting group for the imidazole nitrogen such as Tos or 2,4-dinitrophenyl(DNP), and when Tyr is present, X is hydrogen or a protecting group for the hydroxyl group such as DCB. When Met is present, the sulfur may be protected, if desired, with oxygen.

The selection of a side chain amino protecting group is not critical except that it should must be one which is not removed during deprotection of the α-amino groups during the synthesis. Hence, the α-amino protecting group and the side chain amino protecting group cannot be the same.

X⁷ is NH₂, a protecting group such as an ester or an anchoring bond used in solid phase synthesis for linking to a solid resin support, preferably one represented by the formulae: --NH-benzhydrylamine (BHA) resin support and --NH-paramethylbenzhydrylamine (MBHA) resin support. Cleavage from a BHA or MBHA resin directly gives the CRF analog amide. By employing a methyl-derivative of such a resin, a methyl-substituted amide can be created.

In the formula for the intermediate, at least one of X, X¹, X², X³, X⁴, X⁵ and X⁶ is a protecting group. The particular amino acid chosen for each the R-group determines whether there will also be a protecting group attached as specified hereinbefore and as generally known in the art. In selecting a particular side chain protecting group to be used in the synthesis of the peptides, the following rules are followed: (a) the protecting group should be stable to the reagent and under the reaction conditions selected for removing the α-amino protecting group at each step of the synthesis, (b) the protecting group should retain its protecting properties and not be split off under coupling conditions and (c) the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not alter the peptide chain.

For the acyl group at the N-terminal represented by Y, acetyl, formyl, acrylyl and benzoyl are preferred. For the 1 to 10 amino acid peptide which may be optionally included without adversely affecting the potency, any amino acids may be used, but the L- or D-forms of the naturally occurring amino acids would normally be used.

Thus, the present invention is also considered to provide a process for the manufacture of compounds defined by the Formula (I) comprising (a) forming a peptide having at least one protective group and having the Formula (II) wherein: X, X¹, X², X³, X⁴, X⁵ and X⁶ are each either hydrogen or a protective group, and X⁷ is either a protective group or an anchoring bond to resin support or NH₂ and (b) splitting off the protective group or groups or anchoring bond from said peptide of the Formula (II) and (c) if desired, converting a resulting peptide into a nontoxic addition salt thereof.

When the peptides are prepared by chemical synthesis, they are preferably prepared using solid phase synthesis, such as that described by Merrifield, J. Am. Chem. Soc., 85, p 2149 (1964), although other equivalent chemical syntheses known in the art can also be used as previously mentioned. Solid-phase synthesis is commenced from the C-terminal end of the peptide by coupling a protected α-amino acid to a suitable resin as generally set forth in U.S. Pat. No. 4,244,946 issued Jan. 21, 1981 to Rivier et al., the disclosure of which is incorporated herein by reference. Such a starting material for rCRF analogs can be prepared by attaching α-amino-protected Ile to a BHA resin.

Ile protected by BOC is coupled to the BHA resin using methylene chloride and dimethylformamide (DMF). Following the coupling of BOC-Ile to the resin support, the α-amino protecting group is removed, as by using trifluoroacetic acid(TFA) in methylene chloride, TFA alone or with HCl in dioxane. Preferably 50 volume % TFA in methylene chloride is used with 0-5 weight % 1,2 ethanedithiol. The deprotection is carried out at a temperature between about 0° C. and room temperature. Other standard cleaving reagents and conditions for removal of specific α-amino protecting groups may be used as described in Schroder & Lubke, "The Peptides", 1 pp 72-75 (Academic Press 1965).

After removal of the α-amino protecting group of Ile, the remaining α-amino- and side chain-protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore. As an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor. The selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as coupling reagents are N,N'-dicyclohexyl carbodiimide(DCCI) and N,N'-diisopropyl carbodiimide(DICI).

The activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art. Examples of suitable activating reagents are carbodiimides, such as N,N'-diisopropyl carbodiimide and N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide. Other activating reagents and their use in peptide coupling are described by Schroder and Lubke, supra, in Chapter III and by Kapoor, J. Phar. Sci., 59, pp 1-27 (1970).

Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in about a fourfold excess, and the coupling is carried out in a medium of dimethylformamide(DMF):CH₂ Cl₂ (1:1) or in DMF or CH₂ Cl₂ alone. In instances where the coupling is carried out manually, the success of the coupling reaction at each stage of the synthesis is monitored by the ninhydrin reaction, as described by E. Kaiser et al., Anal. Biochem. 34, 595 (1970). In cases where incomplete coupling occurs, the coupling procedure is repeated before removal of the α-amino protecting group prior to the coupling of the next amino acid. The coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al., Biopolymers, 1978, 17, pp.1927-1938.

After the desired amino acid sequence has been completed, the intermediate peptide is removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride, which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups X², X³, X⁴, X⁵ and X⁶ and the α-amino protecting group X¹ (unless it is an acyl group which is intended to be present in the final peptide), to obtain the peptide. When using hydrogen fluoride for cleaving, anisole or cresole and methylethyl sulfide are included in the reaction vessel as scavengers. When Met is present in the sequence, the BOC protecting group may be cleaved with trifluoroacetic acid(TFA)/ethanedithiol prior to cleaving the peptide from the resin to eliminate S-alkylation.

The following Example sets forth the preferred method for synthesizing CRF analogs by the solid-phase technique.

EXAMPLE I

The synthesis of [Glu²⁹ ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Glu-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂ is conducted in a stepwise manner on a MBHA hydrochloride resin, such as available from Bachem, Inc., having a substitution range of about 0.1 to 0.5 mmoles/gm. resin.

The synthesis is performed on an automatic Beckman 990B peptide synthesizer using a suitable program, preferably as follows:

    ______________________________________                                                                         MIX                                                                            TIMES                                          STEP  REAGENTS AND OPERATIONS   MIN.                                           ______________________________________                                         1     CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                                  3                                              2     Methanol(MeOH) wash-30 ml. (2 times)                                                                     3                                              3     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                  3                                              4     50 percent TFA plus 5 percent 1,2-ethane-                                                                12                                                   dithiol in CH.sub.2 Cl.sub.2 -70 ml. (2 times)                           5     Isopropanol wash-80 ml. (2 times)                                                                        3                                              6     TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml.                                                            5                                                    (2 times)                                                                7     MeOH wash-40 ml. (2 times)                                                                               2                                              8     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                  3                                              9     Boc-amino acid (10 mmoles) in 30 ml. of either                                                           30-300                                               DMF or CH.sub.2 Cl.sub.2, depending upon the                                   solubility of the particular protected                                         amino acid, (1 time) plus DCCI                                                 (10 mmoles) in CH.sub.2 Cl.sub.2                                         ______________________________________                                    

Coupling of BOC-Ile results in the substitution of about 0.35 mmol. Ile per gram of resin. All solvents that are used are carefully degassed, preferably by sparging with an inert gas, e.g. helium or nitrogen, to insure the absence of oxygen that might undesirably oxidize the sulfur of the Met residue.

After deprotection and neutralization, the peptide chain is built step-by-step on the resin. Generally, one to two mmol. of BOC-protected amino acid is methylene chloride is used per gram of resin, plus one equivalent of 2 molar DCCI in methylene chloride, for two hours. When BOC-Arg(Tos) is being coupled, a mixture of 50% DMF and methylene chloride is used. Bzl is used as the hydroxyl side-chain protecting group for Ser and Thr. P-nitrophenyl ester(ONp) is used to activate the carboxyl end of Asn or Gln, and for example, BOC-Asn(ONp) is coupled overnight using one equivalent of HOBt in a 50% mixture of DMF and methylene chloride. The amido group of Asn or Gln is protected by Xan when DCCI coupling is used instead of the active ester method. 2-Cl-Z is used as the protecting group for the Lys side chain. Tos is used to protect the guanidino group of Arg and the imidazole group of His, and the side chain carboxyl group of Glu or Asp is protected by OBzl. At the end of the synthesis, the following composition is obtained BOC-Ser(Bzl)-Glu(OBzl)-Glu(OBzl)-Pro-Pro-Ile-Ser(Bzl)-Leu-Asp(OBzl)-Leu-Thr(Bzl)-Phe-His(Tos)-Leu-Leu-Arg(Tos)-Glu(OBzl)-Val-Leu-Glu(OBzl)-Met-Ala-Arg(Tos)-Ala-Glu(OBzl)-Gln(Xan)-Leu-Ala-Glu(OBzl)-Gln(Xan)-Ala-His(Tos)-Ser(Bzl)-Asn(Xan)-Arg(Tos)-Lys (2-Cl-Z)-Leu-Met-Glu(OBzl)-Ile-Ile-resin support. Xan may have been partially or totally removed by TFA treatment used to deblock the α-amino protecting group.

In order to cleave and deprotect the resulting protected peptide-resin, it is treated with 1.5 ml. anisole, 0.5 ml. of methylethylsulfide and 15 ml. hydrogen fluoride (HF) per gram of peptide-resin, first at -20° C. for 20 min. and then at 0° C. for one-half hour. After elimination of the HF under high vacuum, the resin-peptide is washed alternately with dry diethyl ether and chloroform, and the peptides are then extracted with de-gassed 2N aqueous acetic acid and separated from the resin by filtration.

The peptide is purified by gel permeation followed by semi-preparative HPLC as described in Rivier et al., Peptides: Structure and Biological Function (1979) pp. 125-128, and Rivier et al., J. Chromatography (1983). The chromatographic fractions are carefully monitored by HPLC, and only the fractions showing substantial purity were pooled.

To check whether the precise sequence was achieved, the rCRF analog was hydrolyzed in sealed evacuated tubes containing constant boiling HCl, 3 μl of thioglycol/ml. and 1 nmol of Nle (as an internal standard) for 9 hours at 140° C. Amino acid analyses of the hydrolysates using a Beckman 121 MB amino acid analyzer showed the following amino acid ratios: Asx(1.9), Thr(0.8), Ser(3.1), Glx(9.0), Pro(2.1), Ala(3.8), Val(0.9), Met(1.9), Ile(2.6), Leu(7.0), Phe(0.9), Lys(1.0), His(2.0) and Arg(3.0), which confirmed that the 41-residue peptide structure had been obtained.

EXAMPLE II

The synthetic peptide AHC having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized using a procedure generally as set forth in Example I.

Specific optical rotation of the rCRF peptide, which was synthesized and purified in the foregoing manner, was measured on a Perkin Elmer Model 141 as [α]_(D) ²²° =-24.2°±1.0 (c=1 in 1% acetic acid) (with correction for the presence of H₂ O and TFA) and had a purity of about 95%.

EXAMPLE III

The synthetic peptide AHC and oCRF were examined for their effects on the secretion of ACTH and β-endorphin in vitro and was also in vivo. The potency of synthetic oCRF to stimulate the secretion of ACTH and β-endorphin by cultured rat pituitary cells was measured using the procedure as generally set forth in Endocrinology, 91, 562 (1972). Half-maximal responses were observed at about 65 picomolar concentrations of the peptide AHC, while synthetic oCRF concentrations of about 250 picomolar were needed to achieve this response. The secretory response to maximal (1-5 nM) concentrations of AHC is at a plateau level. In vivo testing is carried out using the general procedure set forth in C. Rivier et al., Science, 218, 377 (1982).

EXAMPLE IV

The peptide [Acetyl-Gly¹, Leu³³, Glu⁴⁰ ]-rCRF having the formula: Ac-Gly-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Leu-Asn-Arg-Lys-Leu-Met-Glu-Glu-Ile-N₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE V

The peptide [des Ser¹ -Glu² -Glu³, Leu³³, Glu⁴⁰ ]-rCRF having the formula: H-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Leu-Asn-Arg-Lys-Leu-Met-Glu-Gln-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE VI

The peptide [Tyr-Ser¹, Glu²⁹ ]-rCRF having the formula: H-Tyr-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Glu-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE VII

The peptide [Thr²², Glu²⁹,40 ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Arg-Ala-Glu-Gln-Leu-Ala-Glu-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Glu-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE VIII

The peptide [Acrylyl-Leu-Gly-Val¹, Ser², Glu⁴⁰ ]-rCRF having the formula: Acr-Leu-Gly-Val-Ser-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Glu-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE IX

The peptide [Glu⁹, Nle²¹, Glu²⁹, Leu³³ ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Glu-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Glu-Gln-Ala-His-Leu-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure to a greater extent than rCRF.

EXAMPLE X

The peptide [Nle⁸, Ser¹¹, Leu³³, Glu⁴⁰ ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Nle-Asp-Leu-Ser-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Leu-Asn-Arg-Lys-Leu-Met-Glu-Glu-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XI

The peptide [Nle²¹, Har²³, Leu³³, Glu⁴⁰, Nle⁴¹ ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Har-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Leu-Asn-Arg-Lys-Leu-Met-Glu-Glu-Nle-NH₂ is synthesized. Testing in accordance with the general procedurre set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XII

The peptide [Benzoyl-Gly¹, des Gln³, Nle¹², Glu²⁹, Arg³⁶ ]-rCRF having the formula: Bz-Gly-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Nle-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Glu-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Met-Glu-Ile-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XIII

The peptide [Acetyl-Pro⁴, Glu²⁹,40, Tyr³⁷ ]-rCRF(4-41) having the formula: Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Glu-Gln-Ala-His-Ser-Asn-Arg-Lys-Tyr-Met-Glu-Glu-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure to a greater extent than rCRF.

EXAMPLE XIV

The peptide [Gln², Orn²³, Glu²⁹, Leu³⁸ ]-rCRF having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Orn-Glu-Gln-Leu-Ala-Glu-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Glu-Ile-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XV

The peptide [Nle²¹, Tyr³², Leu³³, Glu⁴⁰ ]-rCRF having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-Tyr-Leu-Asn-Arg-Lys-Leu-Met-Glu-Glu-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure to a greater extent than rCRF.

EXAMPLE XVI

The peptide [des pGlu¹ -Gly², Ala²¹, Glu²⁸, Met³⁷ ]-sauvagine having the formula: H-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-Ile-Ala-Lys-Gln-Glu-Lys-Glu-Lys-Glu-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Met-Asp-Thr-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XVII

The peptide [Ala²⁰, Arg²¹, Glu²⁸, Ile³⁹,40 ]-sauvagine having the formula: pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-Ala-Arg-Lys-Gln-Glu-Lys-Glu-Lys-Glu-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Ile-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XVIII

The peptide [Leu²⁶, Har³⁵, Met³⁷, Glu³⁹ ]-sauvagine having the formula: pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gln-Glu-Lys-Leu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Har-Leu-Met-Asp-Glu-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XIX

The peptide [Glu⁹, Nle¹⁸, 21 ]-AHC having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Glu-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XX

The peptide [Nle¹⁸, 21 ]-AHC having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXI

The peptide [D-Pro⁴, Nle¹⁸,21 ]-AHC having the formula: H-Ser-Gln-Glu-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXII

The peptide [D-Tyr³, D-Pro⁴, Nle¹⁸, 21 ]-AHC having the formula: H-Ser-Gln-D-Tyr-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXIII

The peptide [Glu²,13,22, Leu¹², Orn²³, Lys²⁶ ]-AHC having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Leu-Glu-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Glu-Orn-Ala-Glu-Lys-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXIV

The synthetic peptide [Ala¹³ ]-AHC having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-Ala-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXV

The peptide [Leu¹², Glu¹³, Tyr³⁷ ]-AHC having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Leu-Glu-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Tyr-Leu-Glu-Glu-Ala-NH₂. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXVI

The peptide [CML¹⁰,14,19,27,33,38, Har³⁶ ]-AHC having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-CML-Thr-Phe-His-CML-Leu-Arg-Glu-Met-CML-Glu-Met-Ala-Lys-Ala-Glu-Gln-CML-Ala-Glu-Gln-Ala-Ala-CML-Asn-Arg-Har-Leu-CML-Glu-Glu-Ala-NH₂. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXVII

The peptide [CML¹⁰,15,27,37,CMA²²,32,41 ]-AHC having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-CML-Thr-Phe-His-Leu-CML-Arg-Glu-Met-Leu-Glu-Met-CMA-Lys-Ala-Glu-Gln-CML-Ala-Glu-Gln-Ala-CMA-Leu-Asn-Arg-Leu-CML-Leu-Glu-Glu-CMA-NH₂. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXVIII

The peptide [D-Tyr³, D-Pro⁴, Nle¹⁸,21 ]-AHC(3-41) having the formula: H-D-Tyr-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it stimulates the secretion of ACTH and β-END-LI to an amount equal to about 470% of oCRF and also causes a very significant lowering of blood pressure.

EXAMPLE XXIX

The peptide [D-Pro⁴, Nle¹⁸,21 ]-AHC(4-41) having the formula: H-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it stimulates the secretion of ACTH and β-END-LI to an amount equal to about 160% of oCRF and also causes a very significant lowering of blood pressure.

EXAMPLE XXX

The peptide [Glu²², Leu¹², Orn²³, Har³⁶ ]-AHC(4-41) having the formula: H-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Leu-His-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Glu-Orn-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Har-Leu-Leu-Glu-Glu-Ala-NH₂. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of bloood pressure.

EXAMPLE XXXI

The peptide [Arg²³,36 ]-oCRF having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Arg-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Leu-Asp-Ile-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it stimulates the secretion of ACTH and β-END-LI to an amount equal to about 370% of oCRF and also causes a very significant lowering of blood pressure.

EXAMPLE XXXII

The peptide [Nle²¹,Glu²⁹ ]-oCRF having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Glu-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXXIII

The peptide [Nle¹⁸,21,Arg²⁸ ]-oCRF having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Thr-Lys-Ala-Asp-Gln-Leu-Arg-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXXIV

The peptide [D-Pro⁴,Asp²² ]-oCRF(4-41) having the formula: H-D-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Asp-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXXV

The peptide [D-Tyr³,CML³³ ]-oCRF(3-41) having the formula: H-D-Tyr-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-CML-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXXVI

The peptide [Nle¹⁸,Tyr³⁷,Glu⁴⁰ ]-oCRF(2-41) having the formula: H-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Tyr-Leu-Asp-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXXVII

The peptide [D-Tyr³, Nle¹⁸,21 ]-AHC(3-41) having the formula: H-D-Tyr-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXXVIII

The peptide [Nle¹⁸,21,Arg²³ ]-AHC(5-41) having the formula: H-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-LI and causes a very significant lowering of blood pressure.

EXAMPLE XXXIX

The peptide [Nle¹⁸,21,Arg³⁶ ]-AHC(6-41) having the formula: H-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Arg-Leu-Leu-Glu-Glu-Ala-NH.sub.2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and β-END-Li and causes a very significant lowering of blood pressure.

EXAMPLE XXXX

The peptide [D-Pro⁴,Nle²¹,38 ]-rCRF(4-41) having the formula: H-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂ is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it stimulates the secretion of ACTH and β-END-LI in an amount greater than oCRF and also causes a very significant lowering of blood pressure.

CRF analogs exhibit such lowering of blood pressure that they may be particularly valuable for the treatment of high blood pressure conditions and also for the treatment of patients who are to undergo certain types of surgery.

CRF profoundly stimulates the pituitary-adrenalcortical axis, and CRF analogs should be useful to stimulate the functions of this axis in some types of patients with low endogenous glucocorticoid production. For example, CRF should be useful in restoring pituitary-adrenal function in patients having received exogenous glucocorticoid therapy whose pituitary-adrenalcortical functions remain suppressed.

Most other regulatory peptides have been found to have effects upon the central nervous system and upon the gastrointestinal tract. Because ACTH and β-END secretion is the "sine qua non" of mammal's response to stress, it was not surprising that CRF has significant effects on the brain as a mediator of the body's stress response. Accordingly, CRF should also find application in modifying the mood, learning and behavior of normal and mentally disordered individuals. Because CRF analogs elevate the levels of ACTH, μ-END, β-lipotropin, other pro-opiomelanocortin gene products and corticosterone, its administration can be used to induce their effects on the brain and its periphery to thereby influence memory, mood, pain appreciation, etc., and more specifically, alertness, depression and/or anxiety. For example, when administered into the ventricles, CRF increases activity and improves learning performance in rats and thus may function as a natural stimulant.

CRF analogs should also be of use for increasing blood flow to the gastrointestinal tract of mammals, particularly humans and other mammals. All CRF related peptides have been shown to dialate the mesenteric vascular bed. Also, oCRF inhibits gastric acid production, and CRF analogs are expected to also be effective in the treatment of gastric ulcers by reducing gastric acid production and/or inhibiting gastrointestinal functions in a mammal.

CRF analogs or the nontoxic addition salts thereof, combined with a pharmaceutically or veterinarily acceptable carrier to form a pharmaceutical composition, may be administered to mammals, including humans, either intravenously, subcutaneously, intramuscularly, percutaneously, e.g. intranasally, intracerebrospinally or orally. The peptides should be at least about 90% pure and preferably should have a purity of at least about 98%; however, lower purities are effective and may well be used with mammals other than humans. This purity means that the intended peptide constitutes the stated weight % of all like peptides and peptide fragments present. Administration to humans may be employed by a physician to lower blood pressure or to stimulate endogenous gluco-corticoid production. The required dosage will vary with the particular condition being treated, with the severity of the condition and with the duration of desired treatment.

These peptides may also be used to evaluate hypothalamic pituitary adrenal functions in mammals with suspected endocrine or central nervous system pathology by suitable administration followed by monitoring body functions. For example, administration may be used as a diagnostic tool to evaluate Cushing's disease and affective disorders, such as depressive illness.

Such peptides are often administered in the form of pharmaceutically or veterinarily acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron, calcium, barium, magnesium, aluminum or the like (which are considered as addition salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tblet may contain a binder, such as tragacanth, corn starch or galatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.

The peptides should be administered under the guidance of a physician, and pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically or veterinarily-acceptable carrier. Usually, the dosage will be from about 1 to about 200 micrograms of the peptide per kilogram of the body weight of the host animal. In some instances, treatment of subjects with these peptides can be carried out in lieu of the administration of ACTH or corticosteroids, in such instances a dosage as low as about 10 ng/Kg of body weight may be employed. As used herein all temperatures are °C. and all ratios are by volume. Percentages of liquid materials are also by volume.

Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto. For example, substitutions and modifications at other positions in the CRF peptide chain can be made in accordance with present or future developments without detracting from the potency of the analogs. It appears important that the amino acid sequence from about positions 6 through 41 or equivalents thereof be present in the synthetic peptide, whereas the remainder of the molecule does not appear as critical. For instance, instead of the simple amide at the C-terminal, a lower alkyl-substituted amide, e.g. methylamide, ethylamide, etc, may be incorporated. Likewise from one to ten additional amino acid residues can be included at the N-terminal without significantly adversely affecting biological potency. Such peptides are considered as equivalents which fall within the scope of the invention.

Various features of the invention are emphasized in the claims which follow. 

What is claimed is:
 1. A composition for lowering the blood pressure of mammals comprising an effective amount of a synthetic CRF peptide or a nontoxic addition salt thereof having the sequence: R₁ -R₂ -R₃ -R₄ -R₅ -Ile-Ser-R₈ -R₉ -leu-R₁₁ -R₁₂ -R₁₃ -leu-leu-Arg-R₁₇ -R₁₈ -R₁₉ -Glu-R₂₁ -R₂₂ -R₂₃ -R₂₄ -R₂₅ -R₂₆ -R₂₇ -R₂₈ -R₂₉ -Gln-ala-R₃₂ -R₃₃ -Asn-Arg-R₃₆ -R₃₇ -R₃₈ -R₃₉ -R₄₀ -R₄₁ wherein R₁ is Ser, D-Ser or des R₁ ; R₂ is Gln, pGlu, Glu, D-pGlu or des R₂ ; R₃ is Glu, Gly, D-Tyr or des R₃ ; R₄ is Pro, D-Pro or des R₄ ; R₅ is Pro or desR₅ ; R₈, R₁₂, R₁₉ and R₂₄ are selected from the group consisting of leu, Ile, ala, Gly, Val, Nle, Phe and Gln; R₉ is Asp or Glu; R₁₁ is Thr or Ser; R₁₃ is His, Tyr or Glu; R₁₇ is Glu or Lys; R₁₈ is Val, Nle or Met; R₂₁ is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R₂₂ is ala, Thr, Asp or Glu; R₂₃ is Arg, Orn, Har or Lys; R₂₅ is Asp or Glu; R₂₆ is Gln, Asn or Lys; R₂₇ is leu, Ile, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R₂₈ is ala, Arg or Lys; R₂₉ is Gln or Glu, R₃₂ is His, Gly, Tyr or ala; R₃₃ is Ser, Asn, leu, Thr or ala; R₃₆ is Lys, Orn, Arg, Har or Leu; R₃₇ is leu or Tyr; R₃₈ is Met or leu; R₃₉ is Glu or Asp; R₄₀ is Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln; R₄₁ is ala, Ile, Gly, Val, Leu, Nle, Phe, Gln or des R₄₁, provided however that R₃ is D-Tyr or R₄ is D-Pro or R₂₈ is Arg or R₂₉ is Glu- or R₄₀ is Glu or R₃₆ is either Arg oor Har or R₃₇ is Tyr, wherein leu is L-leucine or CαCH₃ -L-leucine (CML) and ala is L-alanine or CαCH₃ -L-alanine (CMA); and a pharmaceutically or veterinarily acceptable liquid or solid carrier therefor.
 2. A method for lowering the blood pressure or increasing gastrointestinal blood flow of an animal, which method comprises administering to said animal an effective amount of a composition of claim
 1. 3. A method for reducing gastric acid production or inhibiting gastrointestinal functions in an animal which method comprises administering an effective amount of a composition of claim
 1. 4. Y-R₁ -R₂ -R₃ -R₄ -R₅ -Ile-Ser-R₈ -R₉ -leu-R₁₁ -R₁₂ -R₁₃ -leu-leu-Arg-R₁₇ -R₁₈ -R₁₉ -Glu-R₂₁ -R₂₂ -R₂₃ -R₂₄ -R₂₅ -R₂₆ -R₂₇ -R₂₈ -R₂₉ -Gln-ala-R₃₂ -R₃₃ -Asn-Arg-R₃₆ -R₃₇ -R₃₈ -R₃₉ -R₄₀ -R₄₁ -NH₂ wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R₁ is Ser, D-Ser or des R₁ ; R₂ is Gln, pGlu, Glu, D-pGlu or des R₂ ; R₃ is Glu, Gly, D-Tyr or des R₃ ; R₄ is Pro, D-Pro or des R₄ ; R₅ is Pro or desR₅ ; R₈, R₁₂, R₁₉ and R₂₄ are selected from the group consisting of leu, Ile, ala, Gly, Val, Nle, Phe and Gln; R₉ is Asp or Glu; R₁₁ is Thr or Ser; R₁₃ is His, Tyr or Glu; R₁₇ is Glu or Lys; R₁₈ is Val, Nle or Met; R₂₁ is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R₂₂ is ala, Thr, Asp or Glu; R₂₃ is Arg, Orn, Har or Lys; R₂₅ is Asp or Glu; R₂₆ is Gln, Asn or Lys; R₂₇ is leu, Ile, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R₂₈ is ala, Arg or Lys; R₂₉ is Gln or Glu, R₃₂ is His, Gly, Tyr or ala; R₃₃ is Ser, Asn, leu, Thr or ala; R₃₆ is Lys, Orn, Arg, Har or Leu; R₃₇ is leu or Tyr; R₃₈ is Met or leu; R₃₉ is Glu or Asp; R₄₀ is Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln; R₄₁ is ala, Ile, Gly, Val, Leu, Nle, Phe, Gln or des R₄₁, provided however that R₃ is D-Tyr or R₄ is D-Pro or R₂₈ is Arg or R₂₉ is Glu- or R₄₀ is Glu or R₃₆ is either Arg or Har or R₃₇ is Tyr, wherein leu is L-leucine or CαCH₃ -L-leucine (CML) and ala is L-alanine or CαCH₃ -L-alanine (CMA); or a nontoxic addition salt thereof.
 5. The compound of claim 4 wherein R₃₃ is leu.
 6. The compound of claim 4 wherein R₂₉ is Glu.
 7. The compound of claim 4 where in R₄₀ is Glu.
 8. The compound of claim 4 wherein R₂₈ is Arg.
 9. The compound of claim 4 wherein R₃ is D-Tyr.
 10. The compound of claim 4 wherein R₄ is D-Pro.
 11. The compound of claim 4 wherein R₃₇ is Tyr.
 12. The compound of claim 4 wherein R₃₆ is Arg or Har.
 13. The compound of claim 4 wherein R₂₂ is ala and R₂₇ is Glu.
 14. The compound of claim 4 wherein R₂₄ is ala and R₂₆ is Gln.
 15. The compound of claim 4 having the formula: H-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂.
 16. The compound of claim 4 having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Arg-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Leu-Asp-Ile-Ala-NH₂.
 17. The compound of claim 4 having the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂.
 18. The compound of claim 4 having the formula: H-D-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂.
 19. A method of increasing the secretion of ACTH and corticosteroids or the secretion of β-endorphin, which method comprises administering to a mammal either intraveiously, subcutaneously, intramuscularly, percutaneously, intracerebrospinally or orally an effective amount of the compound of claim
 4. 20. A method of evaulating hypothalamic pituitary adrenal function in mammals with suspected endocrine or central nervous system pathology, which method comprises administering to a mammal either intravenously, subcutaneously, intramuscularly, percutaneously, intracerebrospinally or orally an effective amount of a peptide, or a nontoxic addition salt thereof, having the formula: Y-R₁ -R₂ -R₃ -R₄ -R₅ -Ile-Ser-R₈ -R₉ -leu-R₁₁ -R₁₂ -R₁₃ -leu-leu-Arg-R₁₇ -R₁₈ -R₁₉ -Glu-R₂₁ -R₂₂ -R₂₃ -R₂₄ -R₂₅ -R₂₆ -R₂₇ -R₂₈ -R₂₉ -Gln-ala-R₃₂ -R₃₃ -Asn-Arg-R₃₆ -R₃₇ -R₃₈ -R₃₉ -R₄₀ -R₄₁ -NH₂ wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R₁ is Ser, D-Ser or des R₁ ; R₂ is Gln, pGlu, Glu, D-pGlu or des R₂ ; R₃ is Glu, Gly, D-Tyr or des R₃ ; R₄ is Pro, D-Pro or des R₄ ; R₅ is Pro or desR₅ ; R₈, R₁₂, R₁₉ and R₂₄ are selected from the group consisting of leu, Ile, ala, Gly, Val, Nle, Phe and Gln; R₉ is Asp or Glu; R₁₁ is Thr or Ser; R₁₃ is His, Tyr or Glu; R₁₇ is Glu or Lys; R₁₈ is Val, Nle or Met; R₂₁ is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R₂₂ is ala, Thr, Asp or Glu; R₂₃ is Arg, Orn, Har or Lys; R₂₅ is Asp or Glu; R₂₆ is Gln, Asn or Lys; R₂₇ is leu, Ile, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R₂₈ is ala, Arg or Lys; R₂₉ is Gln or Glu, R₃₂ is His, Gly, Tyr or ala; R₃₃ is Ser, Asn, leu, Thr or ala; R₃₆ is Lys, Orn, Arg, Har or Leu; R₃₇ is leu or Tyr; R₃₈ is Met or leu; R₃₉ is Glu or Asp; R₄₀ is Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln; R₄₁ is ala, Ile, Gly, Val, Leu, Nle, Phe, Gln or des R₄₁, provided however that R₃ is D-Tyr or R₄ is D-Pro or R₂₈ is Arg or R₂₉ is Glu- or R₄₀ is Glu or R₃₆ is either Arg or Har or R₃₇ is Tyr, wherein leu is L-leucine or Cα-CH₃ -L-leucine (CML) and ala is L-alanine or Cα-CH₃ -L-alanine; and thereafter monitoring blood pressure. 